基于量子化学及分子动力学探讨非水相合成OMO型结构脂肪过程中酶催化失活机理及其调控研究

The decrease of lipase activity and exacerbation of acyl migration are “bottle-neck” problems in development of structural lipids. The study of the formation of the transition state intermediate and the microstructure changes of the enzyme can help to elucidate the mechanism of inactivation of the lipase during the reaction. Based on the preliminary study, the project will continue to use camphor seed oil to synthesize OMO-type structured lipid by lipozyme RM IM. The aim of this project focuses on better understanding of the mechanism of lipase inactivation and acyl migration. In the present study, X- ray diffraction，circular dichroism spectroscopy and bioinformatics will be adopted to observe the microstructure change of lipase; the kinetic model of the reactions are established by MATLAB and the thermodynamic parameters are obtained by isothermal titration calorimetry. To further study the mechanism of the lipase inactivation from the point view of molecular level, the theoretical calculation combined quantum chemistry and molecular dynamics (QM / MM) will be utilized. The formation and dissociation of transition state intermediate, the cleavage and reconstruction of substrate ester bond, the electron transfer pathway and energy changes of the substrate will be analyzed by QM/MM. From the perspectives of theory and experiment to observe the microstructure and energy changes of lipase during reaction, the deactivation mechanism of lipase and its regulation mechanism will be elucidated through exploring reaction factors (solvent, substrate ratio, temperature, moisture, time, etc.) - the catalytic site of the enzyme and transition state Intermediate changes – the decrease of enzyme activity and the regularity of acyl migration. We hope that the results of current study could provide a theoretical basis and technical supports in application of lipase synthesis of fats or oils to produce structured lipids in oil industry.

酯酶活力下降迅速及酰基迁移加剧等问题严重制约酶法在结构脂肪合成中应用。从酶微观结构变化及酶与底物形成过渡态中间体角度考虑反应因素对酶活力影响有助于深入阐明酯酶失活机理。本项目在前期研究基础上，利用酶催化樟树籽油合成OMO型结构脂肪为研究对象，围绕反应过程中，酯酶活力下降及酰基迁移加剧等问题，采用X-射线衍射、圆二色谱及生物信息学研究酶微观结构变化，利用MATLAB建立反应动力学模型，等温滴定量热法观察反应热力学参数，并结合量子化学与分子动力学联用（QM/MM）理论，分析反应过渡态中间体形成及解离信息、底物酯键断裂及重构电子转移路径及能量变化，理论与实验相结合，探讨反应因素（溶剂分子、底物比、温度、水分、时间等）— 酶催化位点及过渡态中间体变化 — 酶活力下降及酰基迁移规律性，从微观结构及能量变化角度深入阐明酶失活机理及其调控机制。通过本项目的实施为油脂工业酶法合成结构脂肪提供理论依据。

酯酶活力下降及酰基迁移是脂酶催化合成结构脂中主要瓶颈问题。本项目从酶催化本质上探讨具体反应机理。首先研究了水分在RMIM酯酶合成OMO型结构脂肪过程的作用，观察酰基迁移程度及脂肪酶活力保持情况。确定OMO结构脂肪最大收率为45.65％。分子动力学模拟(MD)中低含水量（5％）水分子可通过氢键稳定酶整个结构，有助于固定酶催化的活性位点，使底物更容易插入活性位点，导致酶活性增加，而较高水分活度或较长反应时间均有利于酰基迁移。进一步使用MD模拟来研究溶剂对结构酯合成中脂肪酶的结构催化特性的影响。均方根偏差（RMSD）分析表明，甲醇，超临界CO2或离子溶剂等对酶的结构造成不稳定性。而己烷（0.239 nm）和甘油三油酸酯（0.188 nm）模拟显示RMSD、均方根涨落（RMSF）和回转半径（Rg）值均较低，且有序二级结构含量较高，表明此两种溶剂可以使酶的结构更加稳定。另外为探讨无酶非催化途径和脂酶催化活性及酰基迁移反应机制，利用B3LYP密度泛函和6-31G(d, p)方法建立无酶非催化途径模型；并根据实验得到脂酶4TGL的晶体结构，建立包含123个原子的脂酶催化途径模型。研究结果显示脂肪酶参与的催化途径为四步反应机理：(1) His235去质子化Ser144，同时Ser144夺取底物1, 2-DA sn-1位置上的羟基质子，实现双质子的转移；(2)底物1, 2-DA sn-1上的氧阴离子亲核攻击sn-2羰基碳，形成五元环；(3) Ser144被His235重新质子化，但Ser144却质子化了底物1, 2-DA sn-2上新形成的羰基氧阴离子，实现双质子的返回；(4) 伴随着质子从sn-2羰基碳转移至sn-1酯氧，一个水分子参与的五元环打开使1, 2-DA变成了1, 3-DA，实现了酰基迁移的过程。其中第4步是整个反应的限速步骤，能垒为18.8 kcal/mol。另外我们还发现脂酶和一个水分子参与的酰基迁移过程中，作为广义酸/碱的脂酶催化三联体（Asp-His-Ser）和“质子穿梭机”的水分子可以协同降低反应所需能垒。酯酶参与催化途径的酰基迁移比分子内的无酶非催化途径低约2倍的能量势垒，这一结果暗示酯酶可降低酰基迁移反应的能垒、加速酰基迁移的速率。通过本项目研究可为深入理解酶促酯交换生产结构脂中酰基迁移现象及对酯酶活力影响提供理论依据。